Suppressing method of iso-citric acid formation in producing citric acid from hydrocarbons by fermentation

ABSTRACT

The present invention relates to a suppressing method of iso-citric acid formation in producing citric acid from hydrocarbons by fermentation. 
     This process is carried out by culturing the microorganisms selected from the group belonging to Candida tropicalis, Candida lipolytica, Candida intermedia and Candida brumptii and their mutants and variants in the culture medium containing paraffinic and olefinic hydrocarbons and their mixture as carbon source under aerobic conditions, wherein specific non-ionic surface active agent is added to said culture medium. 
     The specific non-ionic surface active agent added to said culture medium is selected from the group of sorbitan fatty acid esters and polyoxy-ethylene sorbitan fatty acid esters. The amount of specific surface active agent added to said culture medium is enough from 0.005 to 0.5 percent by weight, preferably from 0.02 to 0.2 percent on the weight basis of said culture medium.

BACKGROUND OF THE INVENTION

The present invention relates to a suppressing method of iso-citric acidformation in producing citric acid from hydrocarbons by fermentation.

It has been well known that citric acid is produced by the assimilationof normal paraffins as carbon source.

Furthermore, we, inventors, have proposed the production process ofcitric acid from normal α-olefins of C₈₋₄₀ as carbon source by culturingthe microorganisms selected from the group of Candida tropicalis,Candida intermedia and Candida brumptii and their mutants and theirvariants, in U.S. Pat. No. 4,180,626.

Iso-citric acid is commonly accumulated in the citric acid fermentationusing hydrocarbons as carbon source. The yield of iso-citric acidaccumulated in the culture medium is often equal to or higher than thatof citric acid, though said yield is somewhat different by the kinds ofmicroorganisms and the cultural conditions.

We have discovered that the iso-citric acid formation in the citric acidproduction from hydrocarbons by ferementation is suppressed by addingthe specific non-ionic surface active agents, such as sorbitan fattyacid esters and polyoxy-ethylene sorbitan fatty acid esters to saidculture medium.

In general, the steric isomers are often produced in organic acidfermentations. For suppressing the formation of isomer and dominatingthe normal compound, some mutants have been commonly used. However, saidmethod requires a great deal of work and cost in search of the mutantsand in examination of the cultural conditions.

In the present invention, the formation of iso-citric acid can besuppressed by adding the specific non-ionic surface active agent to theculture medium, and any mutant is not required. This effect has a greatindustrial significance.

SUMMARY OF THE INVENTION

The main object of this invention is to provide a suppressing method ofiso-citric acid formation in producing citric acid by culturing themicroorganisms which are assimilable to paraffinic and olefinichydrocarbons in the culture medium containing the specific non-ionicsurface active agent under aerobic conditions.

Said object is accomplished by cultivating the microorganisms selectedfrom the group of Candida lipolytica, Candida tropicalis, Candidaintermedia and Candida brumptii and their auxotrophic mutants and theirvariants in the culture medium containing paraffins of C₁₀₋₁₈ andolefins of C₈₋₄₀ as carbon source.

The specific non-ionic surface active gents used in this invention areshown in Table 1.

                                      TABLE 1                                     __________________________________________________________________________     Surface active agents used in this invention                                 Sorbitan fatty acid esters and Polyoxy-ethylene                               sorbitan fatty acid esters                                                    Component     Molecular structure                                             __________________________________________________________________________    Sorbitan monolaurate Sorbitan monopalmitate Sorbitan monostearate             Sorbitan tristearate Sorbitan monooleate Sorbitan trioleate Sorbitan          sesquioleate Sorbitan distearate                                                             ##STR1##                                                       Polyoxy-ethylene sorbitan monolaurate Polyoxy-ethylene sorbitan               monopalmitate Polyoxy-ethylene sorbitan monostearate Polyoxy-ethylene         sorbitan tristearate Polyoxy-ethylene sorbitan monooleate Polyoxy-ethylene     sorbitan trioleate                                                                          ##STR2##                                                       __________________________________________________________________________

The amount of specific surface active agent added to the culture medium,though depending on the kind of said agent is enough from 0.005 to 0.5percent on the weight basis of said medium, preferably 0.02-0.2 percentby weight is enough. Furthermore, it is desirable that said agent isadded to the culture medium during from the initial phase to thelogarithmic growth phase of cultivation, but it may be added to theculture medium at the initial phase of cultivation in case that a smallamount of said agent is used.

In case of producing citric acid from hydrocarbons as carbon source, theamount of citric acid produced and the product ratio of citric acid toisocitric acid are dependent on the kind of carbon source (paraffins orolefins) and the composition of the culture medium, especially the kindof organic nutrients.

Paraffinic and olefinic hydrocarbons and their mixture may be used ascarbon source for the fermentation process in this invention.Especially, olefinic hydrocarbons having carbon number of 8or more arepreferable. Particularly, it is the characeristic feature of thisinvention that olefins of C₁₄₋₄₀ unusable in the present market can beutilized.

Normal α-olefins are desirable olefins, but some iso- and inner-olefinsare allowable to be used with normal α-olefins.

The concentration of said hydrocarbons in the culture medium is from 1to 20 percent on the weight basis of said medium, preferably from 5 to15 percent by weight. Comparing the melting point of olefins with thatof paraffins of the same carbon number, olefins show lower melting pointthan paraffins, and as olefins have better dispersion property thanparaffins of the same carbon number in the culture medium, it isadvantageous to use olefins of C₁₄₋₄₀ in the culture medium as thecarbon source.

As the nitrogen source of the culture medium, inorganic and organicammonium salts, such as ammonium chloride, ammonium acetate, and variousnitrogen compounds may be used individually or in mixture.

Ordinary inorganic salts such as phosphate, sulfate, hydrochloric acidsalts, potassium salts, sodium salts, magnesium salts, iron salts,manganese salts, cupper salts and zinc salts may be used as inorganicnutrients in culture medium. Calcium carbonate and alkali compounds maybe used to regulate pH of said medium. Biotin and thiamine as organicnutrients may be used in a trace amount individually or in theirmixture, and also natural substances such as yeast extract or corn steepliquor containing biotin and thiamine may be used. When biotin andthiamine are used individually, the amount less than 1,000 μg/l ofbiotin or thiamine suffices to increase the production amount of citricacid. Even if the amount more than 1,000 μg/l of biotin or thiamine isused individually, the production amount of citric acid does notincrease. Preferable amount of the organic nutrients is suitable from 50to 100 μg/l. In case where both biotin and thiamine are added in saidmedium, the total amount of biotin and thiamine less than 1,000 μg/l,preferably 50-100 μg/l is enough to cultivate. In the process of thisinvention, α-olefin or mixture of olefins may be used as carbon source.These substrates are in liquid state at fermentation temperature.

DETAILED DESCRIPTION OF THE INVENTION

The effect of addition of specific non-ionic surface active agent to theculture medium in this invention will be explained later. Thecompositions of main culture medium are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        The compositions of culture medium                                                          for Seed                                                                             for production                                           ______________________________________                                        NH.sub.4 Cl     6.5    g     2.0     g                                        Na.sub.2 HPO.sub.4.12H.sub.2 O                                                                1.5    g     --                                               KH.sub.2 PO.sub.4                                                                             3.5    g     0.5     g                                        MgSO.sub.4.7H.sub.2 O                                                                         0.5    g     0.5     g                                        FeSO.sub.4.7H.sub.2 O                                                                         10     mg    10      mg                                       MnSO.sub.4.nH.sub.2 O                                                                         0.1    mg    0.1     mg                                       ZnSO.sub.4.H.sub.2 O                                                                          0.1    mg    0.1     mg                                       CuSO.sub.4.5H.sub.2 O                                                                         5      μg 5       μg                                    Yeast extract   200    mg    --                                               Biotin          100    μg 100     μg                                    Thiamine.HCl    100    μg 100     μg                                    Deionized water 1,000  ml    1,000   ml                                       pH              5.0          5.0                                              ______________________________________                                    

The cultural conditions are shown as follows:

Said microorganisms are cultivated under aerobic conditions. Thefermentation temperature is in the range of 25° and 40° C., preferablyabout 30° C. pH value of said medium is in the range of 3-10, preferably4-6. pH value of said medium is regulated by adding alkalis or saltssuch as sodium carbonate or calcium carbonate. The cultivation isordinarily carried out for 50-150 hrs., preferably 80-100 hrs. Citricacid accumulated in said medium is isolated in the form of calciumcitrate by normal method, for example, by filteration or by centrifugalmethod and then purified by chromatographic or ion exchange method.

Citric acid produced is recovered as follows:

Citric acid produced during the cultivation as described above isrecovered in calcium salt form by filtering said medium usingdiatomaceous earth as a filter aid after conditioning pH to 2.0 withhydrochloric acid. The filter cake is washed with water and the washingwater is combined with the clear filtrate and then heated afterneutralizing the combined filtrate by caustic alkali. The combinedfiltrate is cooled and filtrated under the reduced pressure to recovercalcium citrate. Calcium citrate produced is suspended into about 10times volume of water, and heated in the boiling water for about 30minutes after titrating with 50% of aqueous sulfuric acid solution untilthe filtrate slightly indicates the presence of sulfuric acid withaddition of the aqueous solution of barium chloride to said filtrate. Ifnecessary, the resulting solution is filtered while heating afterdecoloring said filtrate and concentrated under the reduced pressure atthe temperature of 50°-60° C. During concentrating said filtrate, theprecipitated calcium sulfate is filtered and continued to theconcentration until the slightly washy sirup is obtained. When thesirup-like concentrated liquor is allowed to be cooled at 0° C., thecrystalline of citric acid is obtained.

The analysis of citric acid, iso-citric acid and total citric acid wascarried out as follows:

citric acid was recovered from the culture medium as the calcium salt.

The obtained salt was slurried with the distilled water and acidified topH of less than 2 with hydrochloric acid to dissolve all solublematerials and was diluted with the distilled water to the predeterminedvolume.

The amount of citric acid contained in said obtained solution wasquantitatively analyzed according to the colorimetric method using thereagent of pentabromacetone [Protein, Nucleic acid and Enzyme Bd. 2,Page 50 (1957) published by Kyoritsu Publishing Co., Tokyo].

Total citric acid (total amount of citric acid and iso-citric acid) wasquantitatively analyzed according to the modified Saffran method[Journal of Agricultural Chemical Society of Japan Bd. 44 , Page 499(1970)].

The amount of iso-citric acid was calculated by subtracting the amountof citric acid from the total amount of citric acid and iso-citric acid.

Hereinafter are described the examples of the present invention. Themethod of the present invention is not to be limited to the followingexamples.

EXAMPLE 1

The cultivation was carried out as follows:

Candida tropicalisIFO-0589 which had a good assimilability to olefinichydrocarbon was used. n-Tetradecene-1 was used as carbon source in theconcentration of 5 or 7 percent on the weight basis of said culturemedium and polyoxy-ethylene sorbitan monooleate was added to saidculture medium as surface active agent. The amount of said agent wasvaried to 0.02, 0.05 and 0.2 percent on the weight basis of said culturemedium respectively. The seed of the microorganism described above wasincubated in 500 ml Erlenmeyer flask containing 30 ml of said mediumwhich was sterilized at 115° C. for 10 minutes and was allowed to becoold at 30° C. Said seed containing the microorganism belonging toCandida tropicalis was inoculated to said medium in the amount of 4platinum wire loops.

The cultivation of seed was carried out at 30° C. on a reciprocatingshaker operated at 120 oscills/minute with 7 cm strokes for 3 days.After about 16-18 hrs. from the beginning of the cultivation, 1% byweight of freshly sterilized calcium carbonate was added to said mediumto regulate pH of about 5.0.

The production cultivation was carried out in 500 ml Erlenmeyer flaskcontaining 30 ml of said medium and 5 percent of said seed on the volumebasis of said medium for 6 days under the same conditions to those ofthe seed cultivation. 4% by weight of freshly sterilized calciumcarbonate was added to said medium to regulate pH in the range of about5.0. After cultivation, citric acid, iso-citric acid and total citricacid were analyzed according to said analytical method. The results wereindicated in Table 3.

                  TABLE 3                                                         ______________________________________                                        Strain: Candida tropicalis IFO-0589                                           Carbon source: n-Tetradecene-1                                                Amount of addition                                                                        Amount of  Amount of Amount of                                    of surface active                                                                         citric     iso-citric                                                                              total citric                                 agent* (wt %)                                                                             acid (g/l) acid (g/l)                                                                              acid (g/l)                                   ______________________________________                                        Non-addition                                                                              23.7       15.6      39.3                                         0.02        31.0       8.5       39.5                                         0.05        33.0       7.1       40.1                                         0.20        35.0       5.3       40.3                                         ______________________________________                                         *polyoxy-ethylene sorbitan monooleate                                    

EXAMPLE 2

It was clearly indicated in Example 2 that the production ratio ofcitric acid to iso-citric acid was influenced by the kinds of surfaceactive agents. Polyoxy-ethylene sorbitan monolaurate (as A),polyoxyethylene sorbitan monooleate (as B), and sorbitan monooleate (asC) were added as surface active agent to said culture medium inconcentration of 0.05 percent on the weight basis respectively.

As the microorganisms, Candida lipolytica IFO-0746 which had a goodassimilability to n-paraffinic hydrocarbon was used to n-Tetradecane,and Candida tropicalis IFO-0589 which had a good assimilability ton-paraffinic and n-olefinic hydrocarbons was used to n-Tetradecane andn-Tetradecene-1 respectively, n-Tetradecane and n-Tetradecene-1 wereused as carbon source in concentration of 5 or 7 percent on the weightbasis of said culture medium respectively. The cultivations were carriedout under the same procedures as those of Example 1.

The results were indicated in Tables 4-6.

                  TABLE 4                                                         ______________________________________                                        Strain: Candida lipolytica IFO-0746                                           Carbon source: n-Tetradecane                                                            Amount of    Amount of Amount of                                    Kind of surface                                                                         citric       iso-citric                                                                              total citric                                 active agents                                                                           acid (g/l)   acid (g/l)                                                                              acid (g/l)                                   ______________________________________                                        Non-addition                                                                            10.0         6.0       16.0                                         B         15.0         0         15.0                                         C         12.0         0         12.0                                         ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Strain: Candida tropicalis IFO-0589                                           Carbon source: n-Tetradecene-1                                                          Amount of    Amount of Amount of                                    Kind of surface                                                                         citric       iso-citric                                                                              total citric                                 active agents                                                                           acid (g/l)   acid (g/l)                                                                              acid (g/l)                                   ______________________________________                                        Non-addition                                                                            23.7         15.6      39.3                                         B         31.8         0.7       32.5                                         A         29.3         1.4       30.7                                         C         32.8         0         32.8                                         ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Strain: Candida tropicalis IFO-0589                                           Carbon source: n-Tetradecane                                                            Amount of    Amount of Amount of                                    Kind of surface                                                                         citric       iso-citric                                                                              total citric                                 active agents                                                                           acid (g/l)   acid (g/l)                                                                              acid (g/l)                                   ______________________________________                                        Non-addition                                                                            21.0         25.0      46.0                                         C         27.5         12.0      39.5                                         B         27.5         10.0      37.5                                         ______________________________________                                    

As shown in Examples 1 and 2, the suppressing effect on iso-citric acidformation in producing citric acid from hydrocarbons by fermentation wasproved by the addition of the specific non-ionic surface active agent tothe culture medium containing Candida tropicalis and Candida lipolytica.Furthermore in the cultivation of other microorganisms, that is, Candidaintermedia, Candida brumptii, the same suppressing effect on isocitricacid formation was observed.

What is claimed is:
 1. A method for suppressing iso-citric acidformation in producing citric acid from hydrocarbons by fermentation,comprising the steps of:providing microoganisms selected from the groupconsisting of Candida tropicalis, Candida lipolytica, Candida intermediaand Candida brumptii and their mutants and their variants; adding saidmicroorganisms to a culture medium containing olefinic hydrocarbons;further adding to said medium from 0.005 to 0.5 percent on a weightbasis of said culture medium of surface active agent selected from thegroup consisting of polyoxyethylene sorbitan monooleate, polyoxyethylenesorbitan monolaurate and sorbitan monooleate; and culturing saidmicroorganisms under aerobic conditions to produce a mixture of citricacid and isocitric acid, wherein the ratio of citric to isocitric acidis at least 2:1.
 2. A method according to claim 1, wherein the amount ofsaid surface active agent is from about 0.02 to about 0.2 weight percentof said nutrient system.